Dynamics and Mechanism of DNA-Bending Proteins in Binding Site Recognition

Abstract

Dynamics and Mechanism of DNA-Bending Proteins in Binding Site Recognition Many cellular processes involve interactions between proteins and DNA in which proteins recognize and bind to specific sites on the DNA with thousand- or million-fold higher affinities than to random DNA sequences. How these proteins search for and find their specific sites in genomic DNA amidst a large excess (~3 billion) of nonspecific sites remains a puzzle. Many site-specific proteins kink, bend or twist DNA at that site, and undergo concerted conformational rearrangements to accommodate the deformed DNA (‘induced-fit mechanism’). In many cases, the proteins discriminate between specific and nonspecific sites primarily by sensing differences in local DNA deformability (‘indirect readout’), rather than by relying on direct interactions with target nucleotides. How rapidly the deformations occur during target recognition and how they compare with the time that a searching protein spends on a given DNA site before diffusing away remain largely unknown, obscuring our understanding of target recognition mechanisms. Site-specific recognition is expected to be fast or comparable to the protein’s ‘residence time’ per DNA site. Direct observations of proteins undergoing one-dimensional diffusion on nonspecific DNA indicate stepping times (or residence times) per base pair ranging from 50 ns 500 s, considerably shorter than the ~10 ms timescales previously reported for DNA conformational dynamics during binding-site recognition. This posed a puzzle, and suggested that previous studies were likely not resolving key dynamical steps that led to target recognition. I will present recent results on DNA conformational dynamics for three specific protein-DNA complexes: (1) IHF: a prokaryotic architectural protein that recognizes and severely bends specific sites on -phage DNA into a U-turn; (2) XPC: a DNA repair protein that recognizes bulky lesions, unwinds the DNA at that site, and flips out the damaged nucleotides; (3) MutS: another DNA repair protein that recognize mismatches in DNA and sharply kinks the DNA at the mismatched site. All three proteins rely primarily on indirect readout to recognize their DNA target site and therefore must have the ability to sense and discern sequence-dependent DNA deformability while rapidly scanning DNA in search for their target sites. Using nanosecond laser temperature-jump perturbation approach in combination with novel fluorescent probes that enabled protein-DNA dynamics to be measured on timescales of 20 s to > 50 ms, my studies have uncovered previously unresolved steps in the recognition process, including rapid (sub-ms) DNA bending and unwinding that are commensurate with rapid searching while nonspecifically bound, and slower specific recognition steps such as nucleotide flipping or severe DNA bending to form a tight fit. These kinetics measurements help illuminate how a searching protein interrogates DNA deformability and eventually ‘stumbles’ upon its target site, revealing rich multi-step dynamics during this search-interrogation-recognition process.